A major limitation of antibody therapy is its suboptimal delivery to rumor versus normal tissues. An emerging technology is multistep targeting (MST), which can greatly improve the therapeutic index. Tumor cells are first targeted by an antibody-conjugate, which will subsequently be bound by'a cytotoxic ligand. MST is particularly relevant in pediatric cancers where acute toxicity and late effects arc of great concern. We propose to study scFv- streptavidin MST in neuroblastoma (NB), a metastatic solid tumor in children where standard curative therapy does not exist. Our success in expressing the recombinant antibody-conjugate consisting of single chain Fv fragment and streptavidin has made this proposal possible. NB has unique advantages for MST because of the abundance of target antigen GD2, which is homogeneously expressed without antigen modulation or loss after treatment. Preliminary studies have demonstrated substantial improvement in selective delivery of anti-GD2-scFv-streptavidin (scFv-SA) to NB xenografts, scFv-SA MST, using small biotinylated ligands such as DOTA-biotin, achieved a typical cumulative time-activity differential between minor and blood of >50:1. In contrast, using whole IgG as the targeting agent, the ratio was only 4:1. Moreover, peptides and even bovine serum albnmin can be selective1 y targeted by MST. To date, MST efforts have primarily been focused on radioimmunotherapy, without fully exploiting the superior tumor : normal tissue ratio. MST is particularly relevant in tumor models where metastasis in blood and marrow is common, as in ncuroblastoma. We propose to use in vitro models to optimize cytokine targeting by MST to enhance ADCC, as well as to use biotin-anti-lymphocyte antibody as bispecific reagents to redirect NK/NKT cells to enhance tumor cytotoxicity. In vivo validation will be carried out using xenograft tumor models: (1) to study MST radioimmunotherapy using 90Y- DOTA to ablate subcutaneous and metastatic tumors, and (2) MST-targeted IL2 or anti-CD3/anti-CD56 antibodies to enhance and redirect cytotoxic lymphocytes. Before the implementation of any phase I/I1 clinical trial, these studies will provide critical preclinical information on MST tumor therapy directed against neuroblastoma. MST targeted at GD2 will also be applicable to osteosarcoma, brain tumors and small cell lung cancer. We plan to apply these successes to other antigen systems including gp58 which is widely expressed among Ewing's sarcomas, rhabdomyosarcomas, osteosarcomas, neuroblastomas, and brain minors. These are unique opporttmities to exploit MST for delivery of targeted therapies in these pediatric cancers.